The present invention relates to a biologic material, a process for its preparation and the use thereof in tissue grafts.
The loss of cutaneous material due to various causes, traumatic or metabolic for example, can sometimes prove to be very slow-healing. This can be due to metabolic or local circulatory causes, the patient""s poor state of health or to the size of the wound, as in the case of extensive burns. The ineffectiveness of pharmacological therapy has led physicians to resort to reconstructive surgery, using skin grafts from the same patient whenever possible. An important breakthrough in the treatment of such lesions is the use of techniques for in vitro cell culture.
Another problem involved in the preparation of skin substitutes is represented by the supply of fibroblasts to seed onto the biocompatible matrices. Indeed, it is not always easy to isolate fibroblasts from dermal tissues, especially in the case of elderly or severely weakened subjects. One solution to this problem is offered by the mesenchymal cells present in bone marrow tissue. These cells are very active and can be suitably differentiated into various cell lines when placed in the correct conditions. From these stem cells it is possible to obtain differentiated cells such as fibroblasts, adipocytes, myoblasts, osteoblasts, chondrocytes.
J. Rheinwald and H. Green (Cell, 6, 1975, 331-344) were the first to cultivate keratinocytes which could be successfully used to cover skin lesions in clinical practice (G. G. Gallico et al., N. Engl. J. Med., 311, (1984), 448-451). This innovative technique proved to have its limits, however, the most serious being the extreme fragility of the epithelial layer and the very low take rate. To overcome these limitations, dermal derivatives have been constructed on which keratinocytes can be grown. Yannas et al. (Science, 215, (1982), 174-176) used a mixture of collagen and glycosaminoglycans to obtain a reabsorbable porous material to serve as a skin substitute on lesions characterised by the loss of cutaneous substance.
S. Boyce and J. Hansbrough (Surgery, 103 (1988), 421-431) described the use of layers of collagen and glycosaminoglycans as supports on which to grow keratinocytes for subsequent graft.
Another system for the preparation of dermal substitutes is represented by fibroblast cultures on biocompatible three-dimensional matrices based on synthetic or semisynthetic polymers. It is possible to seed and grow fibroblasts on these structures, thus enabling the production of an extracellular matrix similar to that of natural connective tissue.
Some well-known examples of dermal substitutes are:
1) Dermagraft, developed by Advanced Tissue Science (California), in which human fibroblasts are seeded and cultivated on a matrix formed by polylactic, polyglycolic or polygalactoside acid. These fibroblast-populated matrices are subsequently seeded with keratinocytes, to enhance their more xe2x80x9cphysiologicalxe2x80x9d growth;
2) Graft-skin, by Organogenesis Inc. (Boston U.S.A.) composed of a collagen substrate on which heterologous human fibroblasts are seeded;
3) AlloDerm, produced by Life Cell Corp. (Texas, U.S.A.), constituted by human or pig dermis, left intact and stored at a low temperature. Before use, it can be seeded with autologous fibroblasts and keratinocytes and then used for grafting.
Although these products represent good biological supports for in vitro cultures, their in vivo application is somewhat limited, due to immunological reactions against their non-autologous protein components, as well as to the risk of viral contamination.
Lastly, other products deriving from hyaluronic acid are known to be used in skin grafts thanks to their highly biocompatible and biodegradable materials (Benedetti et al., Biomaterials, 14 (1993) 1154-1160; Cortivo R. et al., Biomaterials, 12 (1991) 727-730) and their lack of immunoreactivity. Indeed, as hyaluronic acid is a component of the extracellular matrix it releases completely natural fragments during its degradation in the tissues.
The present invention relates to a biologic material comprising the following two components:
a) an efficient culture of autologous or homologous bone marrow stem cells partially or completely differentiated into cellular lines of a specific connective tissue and further comprising the extracellular matrix produced by said connective tissue cells,
or alternatively
axe2x80x2) the extracellular matrix secreted by:
bone marrow stem cells partially or completely differentiated into a specific connective tissue, or alternatively,
the specific homologous mature connective tissue cells, said extracellular matrix being free from any cellular component, and
b) a three-dimensional biocompatible and biodegradable matrix consisting of a hyaluronic acid derivative.
The present invention further relates to the processes for preparing said biologic material.
When the biologic material according to the present invention contains the component (a) or the component (axe2x80x2) being the extracellular matrix secreted by connective tissue cells coming from partial or complete differentiation of bone marrow stem cells, the process comprises the following steps:
i) isolating said homologous or autologous stem cells from the bone marrow.
ii) transferring said isolated stem cells onto said biocompatible three-dimensional matrix consisting of a hyaluronic acid ester, and
iii) growing and developing said stem cells upon and inside the biomaterials, by dipping the biologic material coming from the preceding step in a culture medium containing also a differentiating factor in case the desired connective tissue cells are different from fibroblasts, thereby obtaining the biologic material containing the component (a),
and optionally
iv) removing the homologous cellular component of (a), by osmotic lysis, thereby obtaining the biologic material containing the above mentioned component (axe2x80x2).
The process for preparing the biologic material according to the present invention containing the component (axe2x80x2) secreted by mature specific connective tissue cells comprises the following steps:
(ixe2x80x2) isolating said mature cells from the specific connective tissue, and growing them under conventional and specific growth condition depending on the specific mature connective cells
(iixe2x80x2) transferring said mature connective tissue cells onto said three dimensional matrix consisting of said hyaluronic acid derivative,
iiixe2x80x2) growing and developing said connective tissue cells upon and inside said three dimensional matrix,
ivxe2x80x2) removing by means of osmotic lysis the cellular components.
The present invention further relates to the use of said biologic material in tissue grafts.